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Coronavirus Hot Paper
A SARS-CoV-2 Spike Binding DNA Aptamer that InhibitsPseudovirus Infection by an RBD-Independent Mechanism**Anton Schmitz+, Anna Weber+, Mehtap Bayin, Stefan Breuers, Volkmar Fieberg,Michael Famulok,* and G�nter Mayer*
Abstract: The receptor binding domain (RBD) of the spikeglycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) bindsto the human angiotensin-converting enzyme 2 (ACE2)representing the initial contact point for leveraging theinfection cascade. We used an automated selection processand identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and doesnot block the interaction of CoV2-S with ACE2. Nevertheless,infection studies revealed potent and specific inhibition ofpseudoviral infection by the aptamer. The present study opensup new vistas in developing SARS-CoV2 infection inhibitors,independent of blocking the ACE2 interaction of the virus, andharnesses aptamers as potential drug candidates and tools todisentangle hitherto inaccessible infection modalities, which isof particular interest in light of the increasing number of escapemutants that are currently being reported.
Introduction
The coronavirus SARS-CoV-2 binds via its spike protein(CoV2-S) to the extracellular domain of the human angio-tensin-converting enzyme 2 (ACE2) initiating the entryprocess into target cells. CoV2-S is a trimeric, highly
glycosylated class I fusion protein. It binds to ACE2 via thereceptor binding domain (RBD) of its S1 subunit.[1] Thetrimeric spike exists in a closed form which does not interactwith ACE2 and in an open form where one RBD is in the so-called “up” conformation exposing the ACE2 binding site.[2,3]
Upon RBD binding to ACE2 the interaction between the S1and S2 subunits is weakened allowing S2 to undergosubstantial structural rearrangements to finally fuse the viruswith the host cell membrane.[2, 3] The important role of theRBD for viral infectivity is underlined by the analyses ofneutralizing antibodies from sera of human re-convalescents,which reveal binding of these antibodies to RBD.[4,5] Con-sequently, almost all published neutralizing antibodies devel-oped for therapeutic use target RBD, including humanizedmonoclonal antibodies,[6] antibodies cloned from human Bcells[7–9] and single-chain camelid antibodies.[10, 11] However,mutations in RBD of CoV2-S can cause RBD-targetedantibodies ineffectual while the virus�s interaction withACE2 remains unchanged or even found improved.[12] Toaddress this limitation, additional inhibitors of viral infectionand a different mode of action, e.g., by targeting otherdomains of CoV2-S are highly desired but of limited avail-ability.
Against this backdrop, we here report on a DNA aptamerwith a different modality of inhibiting viral infection. As theaptamer does not interact with RBD, it does not interfere withthe binding of CoV2-S to ACE2. Regardless, the aptamerinhibits viral infection, exemplified by employing a CoV2-Spseudotyped virus and an ACE2 expressing cell line. Thesefindings demonstrate that viral infection can be inhibitedindependent of targeting RBD and suggest that inhibitioncould be possible despite the virus has already bound to cells.The results open the path to inhibitors of SARS-CoV-2infection with hitherto inaccessible modes of action.
Results
Selection and Characterization of CoV2-S Binding Aptamers
To identify single-stranded (ss)DNA aptamers that bindto CoV2-S we employed an automated selection procedure.[13]
The trimerized His-tagged extracellular domain of CoV2-S,stabilized in the prefusion conformation, was expressed andpurified from HEK293 cells[14, 15] and immobilized for theselection on magnetic beads. After twelve selection cycles(Supporting Figure 1 a) the enriched ssDNA libraries wereanalyzed for improved CoV2-S binding by flow cytometry
[*] A. Schmitz,[+] A. Weber,[+] M. Bayin, S. Breuers, V. Fieberg,M. Famulok, G. MayerLife and Medical Sciences (LIMES), University of BonnGerhard-Domagk-Str.1, 53121 Bonn (Germany)E-mail: [email protected]
A. Schmitz,[+] M. Bayin, V. Fieberg, M. FamulokMax Planck Fellow Chemical BiologyCenter of Advanced European Studies and Research (caesar)Ludwig-Erhard-Allee 2, 53175 Bonn (Germany)
A. Weber,[+] S. Breuers, M. Famulok, G. MayerCenter of Aptamer Research & Development, University of BonnGerhard-Domagk-Str. 1, 53121 Bonn (Germany)
[+] These authors contributed equally to this work.
[**] A previous version of this manuscript has been deposited ona preprint server (https://biorxiv.org/cgi/content/short/2020.12.23.424171v1).
Supporting information and the ORCID identification number(s) forthe author(s) of this article can be found under:https://doi.org/10.1002/anie.202100316.
� 2021 The Authors. Angewandte Chemie International Editionpublished by Wiley-VCH GmbH. This is an open access article underthe terms of the Creative Commons Attribution Non-CommercialLicense, which permits use, distribution and reproduction in anymedium, provided the original work is properly cited and is not usedfor commercial purposes.
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How to cite:International Edition: doi.org/10.1002/anie.202100316German Edition: doi.org/10.1002/ange.202100316
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using cy5-labelled ssDNA and CoV2-S immobilized onmagnetic particles (Figure 1a). These experiments revealedan increased fluorescence signal of the ssDNA from selection
cycle 12 in the presence of CoV2-S (Figure 1a). No inter-action was observed when particles without CoV2-S orparticles modified with His6-Erk2 or His6-dectin-1 were
Figure 1. Selection of DNA aptamers binding to CoV2-S. a) Interaction analysis of the enriched DNA library from selection cycles 1 (R1) and 12(R12) with respect to empty beads, CoV2-S, Erk2 and Dectin. b) Amount of unique sequences in the DNA populations from selection cycles 1–12and the starting library (SL). c) Fraction of sequences in the DNA population from selection cycles 1–12 and the starting library (SL) sharing theindicated copy numbers. d) Frequency of sequences throughout the DNA population from selection cycles 0–12 belonging to the sequencefamilies 8, 13, 22, 29, or 30. See also Supporting Figure 1d. e) Frequency of representative sequences belonging to one of the families from (d).SP1 (Fam 30), SP2 (Fam 29), SP3 (Fam 22), SP4 (Fam 13), SP5-7 (Fam 8). f) Interaction analysis of aptamers SP1–7, the starting library (SL), andDNA from selection cycle 12 (R12) with CoV2-S. SP5sc: scrambled control sequence with identical nucleotides as SP5 but with different primarystructure. g) Sequence motif of family 8 and assignment of aptamers SP5–7. h) Interaction analysis of the scrambled sequence SP5sc andaptamers SP5–7 with CoV2-S, RBD, ACE2, and CoV-S. i) Interaction analysis of SP6 and shortened variants and defined single point mutantsthereof (j). k) Interaction analysis of SP6, SP6.34, and the respective control aptamers SP6C (see supporting Figure 2d) and SP6.34C (j) withCoV2-S in the absence or presence of Mg2+ ions or K+ ions. a,f, h,i, and k: N = 2, mean + /- SD.
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used, indicating specificity of the enriched ssDNA library. Incontrast, the ssDNA library from selection cycle 1 did notshow interaction with the particles, independent of theirmodification state (Figure 1a). The enriched DNA popula-tions were subjected to next-generation sequencing (NGS), inwhich 106 to107 sequences were analyzed per selection cycle(Supporting Figure 1b). This analysis revealed a strongdecrease in the number of unique DNA sequences, startingfrom selection cycle 4 and levelling between 10% to 5% ofunique DNA sequences in selection cycle 7 to 12 (Figure 1b).Likewise, the distribution of nucleotides within the initialrandom region changed significantly throughout the course ofselection, in which guanine is the most frequently enrichednucleotide (Supporting Figure 1c). These data reveal a strongenrichment of DNA sequences, which is further supported bythe occurrence of sequences with high copy numbers, e.g.,> 100 000 per sequence starting from the DNA populationsfrom selection cycle 5 onwards (Figure 1c). Further in-depthpopulation analysis revealed the occurrence of sequencefamilies, termed family 8, 13, 22, 29, and 30 (Figure 1d,Supporting Figure 1d, Supporting Table 1). Whereas thefrequency of sequences belonging to family 8 started toenrich from cycle 8 onwards, all other families were observedin the DNA populations from selection cycles 4 to 6, havingmaximum frequency between selection cycles 7 to 10 anddeclined afterwards (Figure 1d). We chose representativemonoclonal sequences within each family that reflect theenrichment patterns (SP1-7, Figure 1e) and tested themregarding interaction with CoV2-S using flow cytometry.These studies revealed interaction of the family 8 sequencesSP5, SP6, SP7 with CoV2-S (Figure 1 f,g). All other sequencesand a scrambled version of SP5 (SP5sc) as putative non-binding negative control sequence did not interact with thetarget protein (Figure 1 f). SP5, SP6, and SP7 bind with highspecificity to CoV2-S; no binding to the isolated RBD, ACE2or to the spike protein of SARS-CoV (CoV-S) was observed(Figure 1h). Kinetic analysis by surface plasmon resonance(SPR) of the interaction of CoV2-S with 5’-biotinylatedaptamer variants immobilized on streptavidin coated sensorsurfaces show high affinity binding to CoV2-S, with dissoci-ation constants (KD) between 9 and 21 nanomolar (Table 1,Supporting Figure 2a,b). All aptamers revealed comparableKD values at 37 8C vs. 25 8C (Table 1). A qualitative assay[16] todetermine the impact of the 5’-modifications on the aptamersCoV2-S binding properties revealed only minor influence ofthe 5’-cy5-, 5’-biotin-, or 5’-hydroxyl labels (Supporting Fig-
ure 2c). SP5 showed a slightly decreased binding in thehydroxyl state whereas binding of SP6 to CoV2-S was foundto increase by � 50% in the unmodified state as compared tothe 5’-cy5-modified version (Supporting Figure 2c). Theinteraction properties of SP7 appeared to be independent of5’-modifications (Supporting Figure 2c).
SP6 as a representative of the family 8 was chosen forfurther analysis (Figure 1g). Based on secondary structurepredictions (Supporting Figure 2d), the aptamer was initiallytruncated, yielding SP6.51, and analyzed by flow cytometryfor CoV2-S binding. Interestingly, SP6.51 showed stronglyimproved binding compared to the parental SP6 aptamer(Figure 1 i). Further truncation of SP6, yielding variants with45 nucleotides (nt, SP6.45), 41 nt (SP6.41), or 34 nt (SP6.34)maintained the elevated binding properties. When SP6 wastruncated to 30 nt (SP6.30) binding fell back to the level of theoriginal SP6 aptamer whereas the interaction with CoV2-Swas entirely lost by removing additional 11nt (SP6.19, Fig-ure 1 i, Supporting Figure 2d). Moreover, based on thesecondary structure prediction of SP6.34, we investigatedthe interaction of the point mutants of the minimal aptamervariant SP6.34, namely SP6.34A, SP6.34G and SP6.34C withCoV2-S by flow cytometry. These point mutants were chosento either stabilize (SP6.34C) or destabilize (SP6.34A,SP6.34G) the putative apical stem structure (Figure 1 j).However, all point mutants revealed severely diminishedbinding to CoV2-S, whereas binding of SP6.34A was stilldetectable (Figure 1 i), albeit to a much lesser extent than SP6.Mutating the positions equivalent to SP6.34C in the parentalaptamer SP6, yielding SP6C, also abolishes CoV2-S binding(Figure 1k). To conclude the characterization of SP6, theimpact of mono- and divalent ions on CoV2-S binding wasassessed by flow cytometry of both, the parental and minimalvariant of the aptamer. These studies reveal that the bindingof SP6 to CoV2-S is sensitive towards the presence of K+ andstrongly depends on Mg2+-ions (Figure 1k). The binding ofthe parental aptamer SP6 to CoV2-S was maintained in theabsence of K+-ions, whereas the interaction of the minimalvariant SP6.34 was found to be reduced by about 50%compared to its level obtained in PBS (Figure 1k). These dataindicate that K+-ions are most likely required for supportingstructure formation of the aptamer, which is more pro-nounced in the truncated variant than in the parental full-length aptamer, but not essential for CoV2-S binding.
Aptamers Selected for the RBD Do Not Interact with CoV2-S
We also performed automated selection procedures totarget the isolated RBD of Cov2-S (Supporting Figure 3).Conventional automated selection conditions, as appliedtargeting CoV2-S (Figure 1), resulted in strong overamplifi-cation during the PCR step (Supporting Figure 3 a), whichcould be decreased by reducing the amount of target (10%compared to the conventional approach, Supporting Fig-ure 3b) or by adding heparin as a competitor during theincubation step of the selection procedure (Supporting Fig-ure 3c). Interaction analysis of the obtained DNA librariesfrom the selection cycles in which no or very low over-
Table 1: Kinetic properties of the aptamers SP5, SP6 and SP7 measuredby surface plasmon resonance.
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amplification was observed, that is, cycle 6 of the conventionalprocedure (Supporting Figure 3 a), cycle 9 when less targetwas used (Supporting Figure 3 b), and cycle 8 when heparinwas added (Supporting Figure 3c), revealed enrichment ofRBD binding species in all selections (Supporting Figure 3d).However, none of the enriched RBD-binding librariesinteracted with full-length CoV2-S comprising the completeextracellular domain (Supporting Figure 3d). The startinglibrary, used as negative control, neither bound to RBD nor toCoV2-S, whereas the library enriched for CoV2-S (R12CoV2-S, Figure 1 a), used as positive control, showed bindingto CoV2-S as expected (Supporting Figure 3d). Of note,library R12 CoV2-S also revealed interaction with RBD,although to a lesser extent than to CoV2-S (SupportingFigure 3d). Therefore, we decided to use the library R12CoV2-S to conduct three additional selection cycles (cycles13–15) enriching for those species that bind predominantly toRBD instead of other domains of CoV2-S, that are presum-ably targeted by SP5, SP6, and SP7 (Figure 1 h). We againused the conventional selection approach (cycles 13–15,Supporting Figure 3 e) and a selection variant with less(10 %) RBD than in the preceding selection (cycles 13*–15*, Supporting Figure 3e). In both cases overamplificationwas observed from cycle 13/13* on, although less pronouncedas during the de novo selection targeting RBD underpreviously applied selection conditions (Supporting Figure 3).Both enriched libraries (R15/R15*) showed binding to RBDbut no interaction with CoV2-S (Supporting Figure 3 f). Next-generation sequencing of the obtained libraries revealed twostrongly enriched distinct families (Supporting Figure 3g–i,Supporting Tables 2,3). We chose four representative sequen-ces, RBD1-4, and performed interaction analysis. Theseexperiments were found to be in-line with the observationsobtained with the enriched libraries, that is, the sequencesbound to RBD (Supporting Figure 3 j) but not CoV2-S(Supporting Figure 3k). Despite RBD4, which was found atelevated copy numbers in selection cycle 6 of the selectiontargeting CoV2-S but declining thereafter, all RBD bindingsequences only increased in copy numbers when the targetchanged from CoV2-S to RBD in selection cycles 13–15(Supporting Figure 3 l) and 13* to 15* (Supporting Fig-ure 3m). These data indicate that targeting RBD of CoV2-Swith DNA libraries, in our hands, was not productive inyielding aptamers interacting with the full-length extracellu-lar domain of CoV2-S protein in vitro. We analyzed also theinteraction properties of the previously described DNAaptamers RBD C1 and RBD C4, which were selected forbinding to RBD.[17] Unfortunately, in our assay we couldneither observe binding of the aptamers to RBD nor to CoV-2S (Supporting Figure 4). We would like to note that we useddifferent labels on the aptamers and different proteinconstructs, which may explain these conflicting results.
SP6 Inhibits Viral Infection Independent of the Interaction ofCoV2-S with ACE2
We next performed pulldown experiments to furthercharacterize and verify the interaction of SP6 with CoV2-S
(Figure 2a). In these experiments, biotinylated SP6 or SP6Cwere incubated with the respective protein and the complexeswere collected by adding streptavidin coated magnetic beads.After washing, the remaining proteins were analyzed by SDS-PAGE and Coomassie staining of the gel. Aliquots were takenand analyzed prior to the incubation with the magnetic beads(input, Figure 2a), from the supernatant after incubation withthe magnetic beads (unbound, Figure 2 a) and from the bead/aptamer bound fraction (eluate, Figure 2a). SP6 revealedbinding to CoV2-S (Figure 2a, eluate, lane 2) but not toCoV-S (eluate, lane 6) nor to ACE2 (eluate, lane 4) or theunrelated control protein Nek7 (eluate, lane 5). In thisexperiment, SP6C showed weak binding to CoV2-S (Fig-ure 2a, eluate, lane 1). In agreement with the results obtainedby flow cytometry (Figure 1h) binding of SP6 to CoV2-S wasnot reduced even in the presence of a fivefold molar excess ofRBD (Figure 2a, eluate, lane 3).
As SP6 appears not to interact with the RBD of CoV2-S,we investigated whether SP6 has an impact on the interactionof CoV2-S and ACE2. To this end, His-tagged CoV2-S waspulled by Ni-NTA magnetic beads and the co-pulldown ofuntagged ACE2 (ACE2DHis) was analyzed in the presence orabsence of SP6 (Figure 2b). As before, input, unbound andeluate fractions were analyzed by SDS-PAGE and Coomassiestaining. Whereas the interaction between CoV2-S and ACE2was abolished by the RBD-binding control nanobody VHHE[18] (Figure 2b, eluate, lane 4), SP6 did not affect thisinteraction (eluate, lane 3). Densitometric analysis of therespective bands resulted in ACE2:Cov2-S ratios of 0.18 and0.16 in the absence (eluate, lane 1) or presence (eluate, lane 3)of SP6, respectively. To further substantiate this finding,complex formation between CoV2-S-RBD and ACE2 wasmonitored by analytical size exclusion chromatography(Supporting Figure 5). Again, complex formation was pre-vented by VHH E but was unaffected by SP6.
Having shown SP6 interacts with CoV2-S without inter-fering with complex formation with its cellular receptorACE2, we next studied the impact of SP6 on viral infection.To address this question, we used the established VSV-DG*-based pseudotype system[19,20] and generated Cov2-S andVSV-G pseudotyped virus particles. The interaction of SP6with the CoV2-S pseudotyped virus was verified by anenzyme-linked oligonucleotide assay (ELONA).[21] In thisexperiment, the CoV2-S protein or the CoV2-S pseudotypedvirus were captured by a nanobody binding to the RBD ofCoV2-S and after washing the bound protein or pseudovirusparticles were detected by adding biotinylated SP6, strepta-vidin-horse radish peroxidase (HRP) conjugates and itssubstrate 2,2’-Azino-di(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) (Supporting Figure 6). We observed a concentrationdependent increase in signal when SP6 and SP6.34 were usedfor detection, but not when employing SP6C and SP6.34C(Supporting Figure 6a). Likewise, SP6 but not SPC6C de-tected the CoV2-S pseudotyped virus. The VSV-G pseudo-type was not detected demonstrating the specificity of theassay (Figure 2c). Next, ACE2-expressing Vero E6 cells wereinfected with Cov2-S or VSV-G pseudotyped virus particles,which had been pre-incubated with SP6 or SP6C (Figure 2d).Pseudotype particle numbers were adjusted to result in
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Figure 2. RBD-independent inhibition of CoV2-S pseudovirus infection. a) Pulldown analysis of SP6 binding specificity. DST indicates constructslacking the StrepTag. b) Pulldown analysis of CoV2-S ACE2 interaction. DHis indicates lack of His tag. c) ELONA of S protein and SARS-CoV-2-Spseudotype virus. d) SARS-CoV-2-S pseudovirus infection. n = 5, mean + /- SD, *** p<0.001, ** p<0.01, * p<0.05. e) SARS-CoV-2-S pseudovirusbinding. n = 8, mean + /- SD *** p<0.001.
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infection rates between 8 % and 10% for the aptamer-untreated pseudotypes (Supporting Figure 6 b,c). This infec-tion rate was chosen to prevent multiple infections of a singlecell precluding reliable measurements. SP6 showed a concen-tration-dependent reduction of infection of Vero E6 cells bythe CoV2-S pseudotype virus (Figure 2d, Supporting Fig-ure 6b,c). In contrast, the infection of the VSV-G pseudotypewas not affected (Figure 2 d). These results demonstrate thedependence of the inhibitory effect of SP6 on the presence ofCoV2-S on the viral particles and exclude unspecific effectson the infection process of the VSV-~G vector. The presenceof SP6C also led to some reduction of infection which,however, did not reach statistical significance. The seemingdiscrepancy to the lack of binding of SP6C to CoV2-S in thebinding assay (Figure 1k) or the ELONA (Figure 2c) isexplained by the higher concentrations of SP6C used in theinfection assay. In addition, unmodified SP6 (as used in theinfection assay) shows stronger binding to CoV2-S than the 5’-modified versions (Supporting Figure 2c) and this can also beassumed for SP6C. The slight inhibitory effect of SP6C is in-line with its observed weak interaction with CoV2-S in thepulldown assay (Figure 2a).
Whereas ACE2 is the most important receptor for CoV2-S, at least two co-receptors are known to contribute to CoV2-S binding to target cells, heparan sulfate and neuropilin-1.[22,23]
Therefore, we investigated whether SP6 affected binding ofCoV2-S pseudotyped particles to cells even if it did not inhibitCoV2-S binding to ACE2. For this purpose, VSV-DG* waspseudotyped with CoV2-S carrying a HiBiT tag at the C-terminus. Vero E6 cells were incubated with these particlesand bound virus was quantified by NanoBiT reconstitution(Figure 2e). Whereas the known inhibitor heparin[24] reducedbinding of CoV2-S pseudotyped particles, neither SP6 norSP6C had an effect on binding. These data show that SP6reduces pseudovirus infection by interfering with a processoccurring after binding of the pseudovirus to cells.
Discussion
In conclusion, we describe the DNA aptamer SP6 bindingto CoV2-S and with the potential to inhibit SARS-CoV-2infection. A remarkable and unexpected feature of SP6 is thatits inhibitory effect does not result from interfering with theinteraction of CoV2-S with ACE2. This feature distinguishesthe mode of action of SP6 from that of CoV2-S targetingantibodies. Currently, the overwhelming majority of theseantibodies bind to the RBD of CoV2-S[6–11] and prevent ACE2interaction by either directly competing with ACE2 forbinding[6–10] or by stabilizing an ACE2 binding-incompetentconformation.[11] Antibodies not binding to RBD but to theN-terminal domain of CoV2-S have also been shown toprevent interaction of CoV2-S with ACE2 although by an yetunknown mechanism.[10] To our knowledge, neutralizingantibodies targeting the S2 domain have not yet beendescribed. At present, the molecular mechanism by whichSP6 inhibits viral infection is unknown. As the binding ofCoV2-S pseudotypes to cells is not affected, we conclude thata step occurring after binding must be impeded. This could
involve preventing S2’ cleavage or destabilizing the prefusionconformation of the spike protein. The latter mechanism hasbeen shown to lead to viruses bearing spike proteins in thepostfusion conformation and has been reported for anantibody neutralizing SARS-CoV.[25] This antibody, however,binds to the RBD. We anticipate that the elucidation of themechanism by which SP6 inhibits infection will provideinsight into how CoV2-S triggers fusion of the viral and hostcell membranes. While this submission was under review,a related study was published describing aptamers that bind tothe RBD domain of CoV2-S and inhibit pseudoviral infectionby a mechanism distinct to the one of SP6.[26]
There is an increasing number of currently reportedmutations in SARS-CoV-2,[9] among which the most recentexample is the apparently faster spreading lineage VUI-202012/01, also named B.1.1.7.[27] This variant shows severalmutations in the RBD resulting in escape of binding to someantibodies. Since more escape mutations in the RBD can beexpected to further arise in the future, RBD-independentmodalities to prevent infection, as revealed by SP6, are ofrelevance and need to be investigated. Along these lines,testing the ability of SP6 to inhibit isolates of SARS-CoV-2and subsequent in vivo infection studies are next steps tofurther develop and validate the aptamer�s therapeuticpotential.
SP6 might be further optimized to increase potency. Forexample, homo- or heterovalent multimers could be engi-neered by combining SP6 with itself or aptamers (or otherligands) binding to different CoV2-S domains, a strategyemployed previously to gain very potent thrombin inhib-itors.[28, 29] Indeed, di- or trimerization of CoV2-S antibodieshas been shown to increase their potency.[11, 30] The automatedselection process enables the rapid generation of aptamers,for example for mutated proteins of SARS-CoV-2 lineagesthat escape treatment regimens by aptamers, antibodies, orother active pharmaceutical ingredients. Likewise, re-selec-tion strategies to adapt SP6 towards mutations are alsopossible. In addition to these features, aptamers providemeans to develop antigen tests, exemplified by the presentedSP6-based ELONA data. The ease by which aptamers can besynthesized, their low batch-to-batch variations and long shelflives predestines SP6 for various diagnostic and treatmentoptions, e.g., as inhalation spray.
Acknowledgements
This work has been made possible by funds from the FederalMinistry of Education and Research (BMBF, 01KI20154 toM.F. and G.M.) and by the German Research Council (DFG,MA3442/7-1 to G.M.). We thank Dr. F.-I. Schmidt and Dr. P.-A. Kçnig (Core Facility Nanobodies, Medical Faculty, Uni-versity of Bonn) for providing the RBD nanobody, Prof. O.Stemmann (University of Bayreuth) for the Dye488-labelledRBD and Prof. J. Schultze for performing NGS (University ofBonn/DZNE). We are grateful to Dr. G. Zimmer (Institute ofVirology and Immunology, Mittelh�usern, Switzerland) forproviding the VSV-DG* and to Prof. J. S. McLellan (TheUniversity of Texas at Austin, USA) for providing the plasmid
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for CoV2-S expression. Open access funding enabled andorganized by Projekt DEAL.
Conflict of interest
A.S., A.W., S.B., V.F., M.F. and G.M. are listed as inventors ona pending patent application on the aptamers described in thisstudy.
Keywords: antiviral · aptamers · coronavirus · SARS-CoV-2 ·SELEX
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Manuscript received: January 8, 2021Accepted manuscript online: March 8, 2021Version of record online: && &&, &&&&
AngewandteChemieResearch Articles
&&&& www.angewandte.org � 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH Angew. Chem. Int. Ed. 2021, 60, 2 – 9� �
These are not the final page numbers!
Research Articles
Coronavirus
A. Schmitz, A. Weber, M. Bayin,S. Breuers, V. Fieberg, M. Famulok,*G. Mayer* &&&&—&&&&
A SARS-CoV-2 Spike Binding DNAAptamer that Inhibits PseudovirusInfection by an RBD-IndependentMechanism
SP6 is a DNA aptamer binding to theSARS-CoV-2 spike glycoprotein andinhibits pseudovirus infection of cells. Asthe aptamer does not interfere with theCoV-2S ACE2 receptor binding domain, itprovides an RBD-independent mecha-nism of virus inhibition.
AngewandteChemieResearch Articles
&&&&Angew. Chem. Int. Ed. 2021, 60, 2 – 9 � 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH www.angewandte.org
These are not the final page numbers! � �
